Concluding Remarks
Polarized exocytosis is an elegantly designed process requiring the spatial and temporal coordination of numerous proteins functioning in vesicle transport at different stages of the secretory pathway. Although a near complete collection of genes critical for vesicle transport have been discovered, the mechanisms for how these proteins coordinate transport, docking and delivery of secretory vesicles is still mysterious. The goal of my thesis work was to tease out the molecular details and functional significance of the interaction between two of these proteins, Sro7 and the Rab GTPase Sec4.
In Chapter 2, studies were directed at understanding the structural details of the interaction between Sro7 and the Rab GTPase, Sec4. Genetic screens in yeast previously
identified Sro7 to interact with the t-SNARE, Sec9 [47, 88]. More recently, Sro7 was also shown to bind in vitro preferentially to the GTP-locked form of Sec4 [89]. These results and genetic properties consistent with Sro7functioning downstream of Sec4 suggested that Sro7 is a direct effector of Sec4. We utilized the known crystal structure of Sro7 to identify conserved, charged residues on the surface of Sro7 and screened for their involvement in binding to Sec4. These biochemical results combined with in vivo suppression studies and computational modeling identified the Sro7-Sec4 docking interface. Specifically, we found that mutations in Sro7 which disrupt the Sro7-Sec4 interaction also demonstrate a clear deficiency in vivo to overcome defects in Exocyst complex function. Interestingly, our bioinformatics analysis of the sequence variation
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within vertebrate members of the Tomosyn family revealed significant conservation within the region that corresponds with the Sec4-Sro7 binding interface on Sro7. Conversely, analysis of the sequence variation within vertebrate Lgl members revealed that the region corresponding to the Sec4-Sro7 binding pocket is significantly more variable than on Tomosyn. These results suggest a possible conserved Rab GTPase effector function in Tomosyn.
Chapter 3 describes a novel in vitro assay that recapitulates post-Golgi vesicle tethering by Sro7-induced clustering of vesicles. Our lab previously found that overexpression of Sro7 in cells results in the accumulation of large clusters of post-Golgi secretory vesicles in the cell and overall cell lethality [113]. Interestingly, this same phenotype is observed when overexpressing the Exocyst subunit, Sec15, another downstream effector of the Rab GTPase, Sec4 [114].
Vesicle clustering induced by both Sro7 and Sec15 depends on Sec4 function, but is independent of the t-SNARE, Sec9 [113, 114]. Studies in this chapter biochemically reconstruct the
clustering phenotype as an in vitro assay. As demonstrated in vivo, the presence of the Rab GTPase Sec4 on the surface of post-Golgi vesicles is critical for vesicle clustering in the in vitro
system as well. Additionally, Sro7 point mutations designed to destabilize the Sro7 C-terminal autoregulatory tail demonstrate that the conformational status of the tail plays an important role in regulating the activity of Sro7-induced vesicle clustering. The in vitro clustering assay provides a useful new method to analyze how post-Golgi vesicle transport and tethering is mediated by Rab GTPases and their effector proteins.
Our current hypothesis is that Sro7 may function as part of a partially redundant, parallel pathway with the Exocyst tethering complex, downstream of the Sec4 GTPase. As mentioned above, overexpression of either Sro7 or the Exocyst complex component, Sec15, results in a similar phenotype of Rab-dependent, SNARE-independent post-Golgi vesicle clusters in the cell.
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Likewise, both Sro7 and Sec15 are direct effectors of Sec4-GTP and both bind to the t-SNARE, Sec9. Additionally, overexpression of Sro7 suppresses a number of Exocyst complex component mutations and deletions, including exo70Δ, sec5Δ and sec3Δ, and Sro7 has genetic properties consistent with a parallel function [47, 89].
Like SRO7, one additional copy of SEC4 (on CEN) strongly suppresses the temperature sensitivity of the Exocyst mutant, sec15-1 [98]. When we combine sec15-1 with a sro7∆, sro77∆
strain, Sec4 can no longer suppress this lethality, suggesting that functional Sro7 is required in order for Sec4 to bypass defects in the Exocyst complex [data not shown, K. Watson,
unpublished]. Again, this view is consistent with our hypothesis that Sro7 functions in a parallel pathway to the Exocyst complex downstream of Sec4. To test if this function depends on the physical interaction between Sec4 and Sro7, we analyzed the ability of Sec4 to suppress sec15-1
in cells where the only copy of SRO7 is deficient in binding Sec4 (Mutants Sro7-K395E and Sro7-R600E, Chapter 2). In the absence of Sec4-Sro7 interaction, Sec4 fails to suppress sec15-1
lethality, suggesting that this physical interaction is essential for Sec4 to bypass defects in the Exocyst complex [data not shown, K. Watson, unpublished]. We also examined the clustering activity of Sro7 mutants deficient in binding to Sec4 in the in vitro clustering assay [G. Rossi, unpublished]. Interestingly, both purified Sro7 mutants—Sro7-K395E and Sro7-R600E—failed to stimulate vesicle clustering when compared to Wildtype Sro7 [data not shown, G. Rossi, unpublished]. These data strongly suggest that the physical interaction between Sro7 and Sec4 is critical for its ability to cluster and perhaps tether post-Golgi vesicles in the absence of a
functional Exocyst complex.
Despite the fact that structurally, Sro7 does not resemble a CATCHR family tethering complex, it likely has the same functional characteristics of this family, providing specificity to
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vesicle transport as a Rab effector and coordinating vesicle docking with regulated SNARE complex assembly through interaction with SNARE proteins. Future studies will determine which aspects of the Sec4-mediated Sro7 pathway, such as vesicle tethering and SNARE complex assembly, function in parallel to those of the Exocyst complex and perhaps how Sro7 might function as a tethering agent to couple Rab-dependent vesicle tethering with SNARE complex assembly and vesicle fusion.
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